The goal of this reserch is to elucidate the molecular mechanisms of the regulation of glycogen phosphorylase. Three areas of study are proposed: (1) kinetic studies of the "nucleoside site" and its regulatory role; (2) affinity labeling and kinetic studies of the glycogen storage site; and (3) studies of the topography of interaction between phosphorylase and its regulatory enzymes, including the purification and crystallization of phosphorylase kinase and phosphatase catalytic subunits. These investigations will yield information essential to our understanding of the molecular mechanism of phosphorylation and dephosphorylation of the enzymes of glycogen metabolism. This research will take the unique approach of combining biochemical, protein chemical, and collaborative x-ray crystallographic studies to investigate this biochemical/protein structural problem. Of particular interest is the function of the nucleoside site which shows synergism with glucose inhibition of phosphorylase activity and may be involved in regulation of glucose homeostasis by the liver. The functional importance of this site will be investigated by isozyme typing and kinetic studies of phosphorylase from tissues having varied permeabilities to and requirements for exogenous glucose. Studies of polysaccharide binding to phosphorylase will be carried out by chemical synthesis of a specific photo-affinity label for the glycogen storage site. Differential labeling of this site, which binds oligosaccharide twenty-fold more strongly than the active site, will allow the binding constants for glycogen at the active site to be obtained by kinetics. By the use of cleavable chemical cross-linking reagents applied to the complex between phosphorylase and its regulatory enzymes the binding domain for the latter on the surface of phosphorylase will be located and delineated.